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Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick

Fig 3

PK resistance, templating activity and infectivity of PrPSc during sequential unfolding and refolding.

The PK sensitivity of suPrP (A) and rfPrP (B) was assessed after treating the SV fractions of the 6 M urea-treated 263K-brain homogenate without (A) or with a refolding step (B), respectively. ‡ indicates the PK resistance of the non-treated 263K-brain homogenate as a control. (C) For T1Ov-21K and T2Ov-19K, SV fractions corresponding to their suPrP (pool of fractions F2 and F3 from Fig 2B and 2C) and fractions corresponding to rfPrP (fraction F10 from Fig 1B and 1C) were treated with PK. Lane 19K and 21K correspond to non-treated samples digested by PK. (D) Relative templating activity of SV-fractionated suPrP263K (pool of fraction F1 to F3), rf1PrP263K and rf2PrP263K (respectively fractions F9 and F2 in Fig 1C). Fractionated assemblies were serially diluted before being used as a template in the PMCA reaction. The resulting products were analysed for PrPres contents and typical western blot analysis of PMCA product in (E) (n≥9 independent assays for 263K prions). Similar experiments have been done for T2Ov-19K and T1Ov-21K prion strains (F and G and for typical western analysis of PMCA product see S7 Appendix). Positive (+++) and negative (—) detection are indicated using a yellow-to-blue gradient scale. The templating activities of fractions F2 and F9 from untreated 263K assemblies and fractions F5 and F6 from untreated T2Ov-19K and T1Ov-21K, respectively, are shown for comparison. (H) Titration of the templating activity of suPrP263K SEC fractions (see also Fig 1E, green line) after dialysis by PMCA (n = 3 independent assays). The templating activity is expressed as a percentage of that found in a 263K brain homogenate used as standard, as calculated by the ratio of the lowest concentrations resulting in a positive PMCA reaction (red curves). (I) Typical PMCA titration analysed by dot-blotting. Tenfold diluted dialysed SEC fractions corresponding to chromatogram peak (elution volume = 8.3) and for the base of the peak (elution volume = 7.2) were submitted to one round of PMCA and analysed for PrPres content by dot-blotting. The titration was compared to that obtained with 263K infected hamster brain homogenate (263K BH) as reference. (J) Survival of hamster PrP transgenic tg7 mice injected intracerebrally with SV-fractionated suPrP263K (pool of fraction F1 to F3 in red line and pool of fraction F9 to F11 in dash red line), rf1PrP263K (blue line) and rf2PrP263K (green line) assemblies. PrPres263K glycopattern (K) and neuroanatomical deposition (L) in the brains of hamster PrP mice inoculated with rf1PrP263K, rf2PrP263K assemblies compared with 263K prions. The mouse used for suPrP263K analysis was healthy and was euthanized at 200 days post inoculation.

Fig 3